Human serum albumin (HSA) is a protein of 585 amino acids that is responsible for a significant proportion of the osmotic pressure of serum, and also functions as a carrier of endogenous and exogenous ligands. It is used clinically in the treatment of patients with severe burns, shock, or blood loss, and at present is produced commercially by extraction from human blood. The production of recombinant human albumin (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991.
In recent years yeast species have been widely used as a host organisms for the production of heterologous proteins (reviewed by Romanos et al, 1992), including rHA (Sleep et al, 1990, 1991; Fleer et al, 1991). Yeasts are readily amenable to genetic manipulation, can be grown to high cell density on simple media, and as eukaryotes are suitable for production of secreted as well as cytosolic proteins.
When S. cerevisiae is utilised to produce rHA, the major secreted protein is mature 67 kDa albumin. However, a 45 kDa N-terminal fragment of rHA is also observed (Sleep et al, 1990). A similar fragment is obtained when rHA is expressed in Kluyveromyces sp. (Fleer et al, 1991) and Pichia pastoris (EP 510 693). The fragment has the same N-terminal amino acid sequence as mature rHA, but the carboxy terminus is heterogeneous and occurs between Phe.sup.403 and Val.sup.409 with the most common termini being Leu.sup.407 and Val.sup.409 Geisow et al, 1991), as shown below.
.dwnarw. .dwnarw. -Phe-Gln-Asn-Ala-Leu-Leu-Val-Arg-Tyr-Thr-Lys-Lys-Val-Pro-Gln- (SEQ ID NO:16) 405 410 415
The amount of fragment produced, as a percentage of total rHA secreted, varies with both the strain and the secretion leader sequence utilised, but is never reduced to zero (Sleep et al, 1990). We have also found that the amount of fragment produced in high cell density fermentation (75-100 g/L cell dry weight) is approximately five times higher than in shake flask cultures.
The 45 kDa albumin fragment is not observed in serum-derived human serum albumin (HSA), and its presence as non-nature-identical material in the recombinant product is undesirable. The problem addressed by the present invention is to reduce the amount of the 45 kDa fragment in the product. The simplest and most obvious approach would have been to have purified it away from the full length albumin, as proposed by Gist-brocades in EP 524 681 (see especially page 4, lines 17-22). However, we have chosen a different approach, namely to try to avoid its production in the first place.
Sleep et al (1990) postulated that rHA fragment is produced within the cell and is not the result of extra-cellular proteolysis. These authors codon-optimised the HSA cDNA from Glu.sup.382 to Ser.sup.419 but this had no effect on production of rHA fragment. They noted that a potential Kex2p processing site in the rHA amino acid sequence, Lys.sup.413 Lys.sup.414, is in close proximity to the heterogeneous carboxy terminus of the fragment, but neither use of a kex2 host strain (ie a strain harboring a mutation in the KEX2 gene such that it does not produce the Kex2p protease), nor removal of the potential cleavage site by site-directed mutagenesis of the codon for Lys.sup.414, resulted in reduction in the amount of the fragment.
There is a vast array of yeast proteases which could, in principle, be degrading a desired protein product, including (in S. cerevisiae) yscA, yscB, yscY, yscS, other vacuolar proteinases, yscD, yscE, yscF (equivalent to kex2p), ysc.alpha., yscIV, yscG, yscH, yscJ, yscE and kex1.
Bourbonnais et al (1991) described an S. cerevisiae endoprotease activity specific for monobasic sites, an example of which (Arg.sup.410) exists in this region of albumin. This activity was later found to be attributable to yeast aspartyl protease 3 (Yap3) (Bourbonnais et al, 1993), an enzyme which was originally described by Egel-Mitani et al (1990) as an endoprotease similar to Kex2p in specificity, in that it cleaved at paired basic residues. Further work suggested that Yap3p is able to cleave monobasic sites and between, and C-terminal to, pairs of basic residues, but that cleavage at both types of sites is dependent on the sequence context (Azaryan et al, 1993; Cawley et al, 1993).
As already discussed, the region of the C-terminus of rHA fragment contains both a monobasic (Arg.sup.410) and a dibasic site (Lys.sup.413 Lys.sup.414). However, even though a Kex2p-like proteolytic activity is present in human cells and is responsible for cleavage of the pro sequence of HSA C-terminal to a pair of arginine residues, the fragment discussed above is not known to be produced in humans. This indicates that the basic residues Arg.sup.410, Lys.sup.413 and Lys.sup.414 are not recognised by this Kex2p-like protease, in turn suggesting that this region of the molecule may not be accessible to proteases in the secretory pathway. Thus, the Yap3p protease could not have been predicted to be responsible for the production of the 45 kDa fragment. In addition, Egel-Mitani et al (1990 Yeast 6, 127-137) had shown Yap3p to be similar to Kex2p in cleaving the MF.alpha. propheromone. Since removal of the Kex2p function alone does not reduce the amount of the fragment produced, there was no reason to suppose that removal of the Yap3p function would be beneficial. Indeed, Bourbonnais et al (1993) showed that yap3 strains had a decreased ability to process prosomatostatin, and therefore taught away from using yap3 strains in the production of heterologous proteins.